Project Abstract Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a vector-borne parasitic disease caused by Trypanosoma brucei infections. It threatens over 60 million people in 36 countries of sub-Saharan Africa where medical resources are very limited. There are no satisfactory drugs for the treatment thus far. Trypanosome infections are now referred to as ?most neglected diseases?, and need more research efforts. The dynamics of microtubules is essential for T. brucei cell division and its flagellar motility. Therefore, tubulin is a promising target of anti-trypanosome agents, since compounds interfering with microtubule dynamics can suppress both cell division and movement. Importantly, trypanosomal and mammalian tubulins have structural differences, allowing development of selective inhibitors with minimal toxicity. On the other hand, the tubulin structures in all disease-causing Kinetoplastids are nearly identical, suggesting that our tubulin inhibitors have the potential to suppress other Kinetoplastid infections. We have identified several drug candidates that selectively decreased trypanosome cell viability. Via oral gavage, these compounds significantly inhibited trypanosomal replication in infected mice. However, the potency of the compounds was not strong enough to clear up the infection completely. Nevertheless, the compounds are good leads for further optimization to generate more effective anti-trypanosome agents. We hypothesize that introducing functional groups that can be recognized by the P2 transporter on trypanosome cell surface will enhance uptake of tubulin inhibitors, which will improve their efficacy and selectivity. To test this hypothesis, we will focus on three research aims listed below: 1) Structurally optimize the tubulin inhibitors to improve the trypanosome cell uptake. We will introduce purine mimetic and imidamide moieties into our tubulin inhibitors to improve their cell uptake, as trypanosomes depends on their mammalian host for purines. We will also test if our compounds can inhibit T. cruzi growth due to the great similarity among tubulin homologues of all trypanosomes. 2) Investigate the mechanisms and in vivo potency of the selected compounds. We expect our tubulin inhibitors block trypanosomal tubulin polymerization, which will be tested. In addition, we will determine if our new compounds show improved in vivo activities, since the uptake of these compounds by trypanosomes is enhanced. 3) Determine if our drug candidates can cross the blood-brain barrier. Our drug candidates showed relatively high hydrophobicity and no ionization in normal physiological condition, suggesting that these compounds are likely able to cross the blood-brain barrier. We will determine the drug concentrations in blood and brain tissue after administering to trypanosome-infected mice.